Dongchun Liang, Aijun Zuo, Hui Shao, Mingjiazi Chen, Henry J. Kaplan and Deming Sun J Immunol published online 3 November 2014 http://www.jimmunol.org/content/early/2014/11/01/jimmun ol.1401959

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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Anti-Inflammatory or Proinflammatory Effect of an Adenosine Receptor Agonist on the Th17 Autoimmune Response Is Inflammatory Environment−Dependent

Published November 3, 2014, doi:10.4049/jimmunol.1401959 The Journal of Immunology

Anti-Inflammatory or Proinflammatory Effect of an Adenosine Receptor Agonist on the Th17 Autoimmune Response Is Inflammatory Environment–Dependent

Adenosine is a key endogenous signaling molecule that regulates a wide range of physiological functions, including immune system function and inflammation. Studies have shown that adenosine receptor (AR) agonists can be either anti-inflammatory or proinflammatory in immune responses and in inflammation, and the clarification of the mechanisms causing these opposing effects should provide a better guide for therapeutic intervention. Whereas previous studies mostly examined the effects of AR agonists on Th1type immune responses, in this study, we compared their effect on Th17 and Th1 autoimmune responses in experimental autoimmune uveitis, a mouse model of human uveitis induced by immunization with the human interphotoreceptor retinoidbinding protein peptides 1–20. We showed that injection of mice with a nonselective AR agonist, 59-N-ethylcarboxamidoadenosine (NECA), at an early stage after immunization had an inhibitory effect on both Th1 and Th17 responses, whereas injection of the same amount of NECA at a late stage inhibited the Th1 response but had an enhancing effect on the Th17 response. We also showed that the effects of NECA on Th1 and Th17 responses were completely dissociated, that the enhancing effect of NECA on Th17 responses was modulated by gd T cells, and that the response of gd T cells to NECA was determined by their activation status. We conclude that the inflammatory environment has a strong impact on converting the effect of AR agonist on the Th17 autoimmune response from anti-inflammatory to proinflammatory. Our observation should help in the designing of better ARtargeted therapies. The Journal of Immunology, 2014, 193: 000–000.

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revious studies have shown that the appropriate generation and clearance of extracellular accumulated adenosine in inflammation are critical in limiting tissue pathology (1–4). Experimental studies have shown that adenosine receptor (AR) agonists and antagonists are promising pharmacological modulators of disease-associated inflammation and immune responses (5–10), but it has been difficult to achieve reproducible beneficial effects because of a lack of knowledge of how adenosine exerts anti-inflammatory or proinflammatory effects (11–13). Previous studies have proposed that the pro-inflammatory and antiinflammatory effects of adenosine are produced by activation of different ARs. For example, the suppressive effects of adenosine are mainly mediated by A2A receptor (A2AR) signaling (14–16), whereas A2B receptor (A2BR) signaling mostly enhances immune responses (13, 17–20). However, this cannot explain the observations that A2AR2/2 mice show increased susceptibility to autoimmune disease and that administration of an A2AR antagonist to mice with actively induced experimental auto*Doheny Eye Institute, University of California, Los Angeles, Los Angeles, CA 90033; †Department of Ophthalmology, University of California, Los Angeles, Los Angeles, CA 90033; and ‡Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, KY 40202 Received for publication August 4, 2014. Accepted for publication October 2, 2014. This work was supported by National Institutes of Health Grants EY0022403, EY018827, and EY003040. Address correspondence and reprint requests to Dongchun Liang, Department of Ophthalmology, University of California Los Angeles, 1450 San Pablo Street, Los Angeles, CA 90033. E-mail address: [email protected] Abbreviations used in this article: A2AR, A2A receptor; A2BR, A2B receptor; AR, adenosine receptor; EAE, experimental autoimmune encephalomyelitis; EAU, experimental autoimmune uveitis; IRBP, interphotoreceptor retinoid–binding protein; LDA, limiting dilution assay; NECA, 59-N-ethylcarboxamidoadenosine. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401959

immune encephalomyelitis (EAE) inhibits, rather than enhances, disease development (21), implying that whether an AR agonist is anti-inflammatory or proinflammatory is sophisticatedly regulated. To identify factors that modulate or convert the enhancing and inhibiting effects of AR agonists, we asked whether T cell subsets at different degrees of activation respond differently to the same AR agonist and whether environmental factors modulate the antiinflammatory and proinflammatory effects of an AR agonist. Because previous studies examining the effect of AR agonists on immune responses have mainly looked at the IFN-g–producing (or Th1) cell response, and because our ongoing study of the regulation of the Th17 autoimmune response in experimental autoimmune uveitis (EAU) showed that gd T cells have a strong regulatory effect on Th17 autoreactive T cells (22–25) and that these cells express high levels of ARs (26), we asked whether AR agonists affect the Th1 and Th17 responses differently and whether the regulation of the Th17 autoimmune response by AR agonists is associated with the regulatory activity of gd T cells in the Th17 response. In this study, using a mouse model of uveitis in which B6 mice are immunized with the human interphotoreceptor retinoid–binding protein (IRBP) peptide IRBP1–20 to induce EAU, thus promoting the in vivo activation of Th1 and Th17 autoreactive T cells (22, 25, 27, 28), we showed that, although AR agonists always had an inhibitory effect on the Th1 autoimmune response, their effect on the Th17 autoimmune response could be either inhibitory or enhancing, depending on both environmental conditions and the activation status of the T cells. A single early injection of the immunized mice (days 0–5 after immunization with IRBP1–20) with an AR agonist had a suppressive effect on the Th17 response, whereas treatment at a later date (days 7–10 postimmunization), when inflammation had already been initiated, had an enhancing effect on the Th17 response. Mechanistic studies showed that the

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Dongchun Liang,*,† Aijun Zuo,*,† Hui Shao,‡ Mingjiazi Chen,*,† Henry J. Kaplan,‡ and Deming Sun*,†

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ENVIRONMENTAL FACTORS MODULATE THE EFFECT OF ADENOSINE

enhancing effect of an AR agonist required the presence of gd T cells and was greatly diminished when the gd T cells were functionally deficient. Moreover, an enhancing effect was only seen for Th17 responses. Our observation that the proinflammatory and anti-inflammatory effects of an AR agonist can be converted by environmental factors implies that successful ARtargeting treatments or immunomodulation requires monitoring of existing environmental conditions.

All animal studies conformed to the Association for Research in Vision and Ophthalmology statement on the use of animals in Ophthalmic and Vision Research. Institutional approval was obtained from the Institutional Animal Care and Use Committee of the Doheny Eye Institute, University of Southern California, and institutional guidelines regarding animal experimentation were followed.

Animals and reagents Female C57BL/6 (B6) and TCR-d2/2 mice on the B6 background, purchased from The Jackson Laboratory (Bar Harbor, ME), were housed and maintained in the animal facilities of the University of Southern California. Recombinant murine IL-1, IL-7, and IL-23 were purchased from R&D Systems (Minneapolis, MN). FITC- or PE-conjugated Abs against the mouse ab TCR, gd TCR, IL-17, IFN-g, or CD44 and isotype control Abs were purchased from Biolegend (San Diego, CA). The nonselective AR agonist 59-N-ethylcarboxamidoadenosine (NECA), the selective A2AR agonist 2-p-(2-carboxyethyl) phenethylamino-NECA (CGS21680), and the selective A2BR agonist (BAY 60-6538) were purchased from SigmaAldrich (St. Louis, MO).

Immunization and NECA treatment EAU was induced in B6 mice by s.c. injection of 200 ml emulsion containing 200 mg human IRBP 1–20 (Sigma-Aldrich) in CFA (Difco,

T cell preparation ab and gd T cells were purified from B6 mice immunized with IRBP1–20 as described previously (22, 25, 28). Nylon wool-enriched splenic T cells were incubated sequentially for 10 min at 4˚C with FITC-conjugated antimouse gd TCR or ab TCR Abs and for 15 min at 4˚C with anti-FITC Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany); then the cells were separated into bound and nonbound fractions on an autoMACS separator column (Miltenyi Biotec). The purity of the isolated cells, determined by flow cytometric analysis using PE-conjugated Abs against ab or gd T cells, was .95%.

Assessment of Th1 and Th17 polarized responses Responder T cells (3 3 106) prepared from IRBP1–20–immunized B6 or TCR-d2/2 mice were cocultured for 48 h with IRBP1–20 (10 mg/ml) and irradiated spleen cells (2 3 106/well) as APCs in a 12-well plate under either Th17 polarized conditions (culture medium supplemented with 10 ng/ml IL-23) or Th1 polarized conditions (culture medium supplemented with 10 ng/ml IL-12); then IL-17 and IFN-g levels in the culture medium were measured using ELISA kits (R&D Systems), and the number of Ag-specific T cells expressing IL-17 or IFN-g was determined by intracellular staining followed by FACS analysis.

Cytoplasmic staining After 5 d of in vitro stimulation of the in vivo primed T cells with the immunizing Ag and APCs, activated T cells were separated using Ficoll gradient centrifugation and stimulated in vitro for 4 h with 50 ng/ml PMA, 1 mg/ml ionomycin, and 1 mg/ml brefeldin A (Sigma-Aldrich). The cells were then fixed, permeabilized overnight with Cytofix/Cytoperm buffer

FIGURE 1. NECA treatment can either suppress or enhance the Th17 response in mouse EAU. Groups of B6 mice were immunized with IRBP1–20/CFA alone or were also injected with NECA (100 ng/mouse) either on the day of immunization (early treatment) or 7 d after immunization (late treatment). All the mice were euthanized 13 d postimmunization, sera were collected, and T cells from the draining lymph nodes and spleens were separated, counted, and subjected to in vitro stimulation with an optimal dose of immunizing peptide (10 mg/ml) and APCs (irradiated spleen cells) in medium supplemented with 10 ng/ml IL-23; then the activated T cells were separated on a Ficoll gradient and subjected to various analyses. Results shown [except in (B)] are the mean 6 SD for one study using four mice, and the experiment was repeated four to five times with similar results. (A) Serum IL-17 levels were measured by ELISA. (B) Cytoplasmic staining assessing the percentage of IL-17+ cells among the in vitro Ag-stimulated IRBP-specific T cells. After 5 d of in vitro stimulation, the activated T cells were treated with PMA, ionomycin, and brefeldin, then were intracellularly stained with PE-conjugated anti-abTCR Abs and FITC-conjugated anti–IL-17 Abs, followed by FACS analysis. (C) Percentage of IL-17+ cells in the ab T cells after in vitro stimulation of in vivo primed T cells with the immunizing peptide IRBP1–20. (D) IL-17 levels in the supernatant of in vitro cultured T cells after exposure to immunizing Ag and APCs. (E) LDA results. Responder T cell frequencies were evaluated by LDA as detailed in Materials and Methods. **p , 0.01.

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Materials and Methods

Detroit, MI) at six spots at the tail base and on the flank, and by i.p. injection with 300 ng pertussis toxin, as described previously (23, 25, 28). For NECA treatment, immunized B6 mice received a single i.p. injection of NECA (5 mg/kg) on different days after immunization; the day of injection was day 0 (early) or 7 (late) postimmunization. Controls were injected with vehicle only.

The Journal of Immunology (eBioscience, San Diego, CA), and intracellularly stained with Abs against IFN-g or IL-17 and analyzed on a FACSCalibur.

Immunofluorescence flow cytometry Aliquots of 2 3 105 cells were double-stained with combinations of FITCor PE-conjugated mAbs. Data collection and analysis were performed on a FACSCalibur flow cytometer using CellQuest software.

Limiting dilution analysis

Induction of EAU by cell transfer For induction of EAU by adoptive transfer, T cells were isolated from lymph node and spleen cells on days 12–14 postimmunization (optimal time for proliferation and cytokine responses) and stimulated for 48 h with 10 mg/ml IRBP1–20 in the presence of irradiated syngeneic APCs; then activated T cell blasts were separated by Ficoll gradient centrifugation and transferred into B6 mice (1.5 3 106 activated cells/mouse).

Scoring of EAU

Results Whether an AR agonist has an anti-inflammatory or proinflammatory effect in mice with actively induced EAU depends on whether it is administered before or after onset of inflammation To examine the effect of an AR agonist on the Th17 autoimmune response and whether this was affected by the time of injection of the agonist, we immunized B6 mice with the uveitogenic peptide IRBP1–20 in CFA, then randomly divided them into three groups (n = 4). One group received an i.p. injection of 100 ng/mouse NECA, a nonselective AR agonist (13) that binds to both A2ARs and A2BRs, during the early stage after IRBP1–20 immunization (days 0–5); another group was treated with NECA 7–10 d after immunization; and the third group was left untreated. A kinetic study of the effects of NECA administration at different time points showed a change in effect from anti-inflammatory to proinflammatory at days 6–7 (data not shown); therefore, in subsequent studies, two representative time points were used, with the agonist being injected on day 0 (during immunization), designated as “early treatment,” or on day 7 after immunization, designated as “late treatment.” At day 13 after immunization (the time point at which the highest T cell response is seen), serum cytokine levels were measured and responder T cells from the spleens and draining lymph nodes were subjected to in vitro stimulation with the immunizing peptide (10 mg/ml) and APCs (irradiated spleen cells) under culture conditions that favor Th17 autoreactive T cell expansion (medium containing 10 ng/ml IL-23) (25, 29, 34), and the activated T cell blasts separated by Ficoll gradient centrifugation and stained intracellularly with FITC-labeled anti–IL-17 Abs. Fig. 1 shows that the responses of T cells from mice that received NECA treatment differed greatly from those of T cells from untreated

The mice were examined three times a week for clinical signs of EAU by indirect funduscopy. The pupils were dilated using 0.5% tropicamide and 1.25% phenylephrine hydrochloride ophthalmic solutions, and funduscopic grading of disease was performed using the scoring system described previously (32). For histopathological evaluation, whole eyes were collected at the end of the experiment and immersed for 1 h in 4% glutaraldehyde in phosphate buffer, pH 7.4, then transferred to 10% formaldehyde in phosphate buffer until processed. The fixed and dehydrated tissues were embedded in methacrylate, then 5-mm sections were cut through the pupillary-optic nerve plane and stained with H&E. Presence or absence of disease was evaluated blind by examining six sections cut at different levels for each eye. Disease was graded pathologically based on cellular infiltration and structural changes (33).

Assessment of the gd T cell response to AR agonists gd T cells were isolated from naive or immunized B6 mice using a MACS column, then 5 3 104/well separated gd T cells were cultured for 48 h in 96-well plates in medium with or without NECA (100 ng/ml), and the supernatants were collected for assessment of IL-17 levels.

Assessment of the enhancing effect of gdTCR+ T cells on Th17 autoreactive T cells ab T cells (1 3 106) from immunized TCR-d2/2 mice were stimulated with the immunizing peptide in 24-well plates for 5 d in the presence or absence of gd T cells (5% of total cell number) isolated from immunized B6 mice that had either been left untreated or had been incubated for 48 h with 100 nM NECA; then the proliferating cells were separated on a Ficoll gradient, intracellularly stained with anti–IL-17 Abs, and subjected to FACS analysis.

Statistical analysis Experiments were repeated four to five times. Experimental groups were typically composed of four mice, and the figures show the data from a representative experiment. The statistical significance of differences between the values for different groups was examined using the two-tailed Student t test.

FIGURE 2. Pathogenic activity of IRBP-specific T cells isolated from immunized mice with or without early or late injection of NECA. Three groups of donor mice were immunized with a pathogenic dose of IRBP/ CFA with or without early or late injection of NECA (100 ng/mouse); then T cells from draining lymph nodes and spleens were separated and subjected to in vitro stimulation with IRBP peptide and syngeneic APCs in medium supplemented with 10 ng/ml IL-23. Activated T cells (1.5 3 106) were then adoptively transferred by i.p. injection into naive B6 mice and EAU scored. Results shown are the mean 6 SD for one study using four mice and the experiment was repeated three times with similar results. (A) Number of IRBP-specific T cells isolated from spleen and draining lymph nodes per IRBP-immunized mouse in each group. (B) EAU in the recipients of IRBP-specific T cells evaluated by funduscopy every 5 d or by pathological examination of the eye 10 d after cell transfer (original magnification 3200, H&E staining) (C).

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B6 and TCR-d2/2 mice immunized with IRBP1–20/CFA were left untreated or were injected with NECA; then the spleen and draining lymph nodes were removed 13 d after immunization and pooled, and a single-cell suspension prepared. T cells were enriched by MACS column purification and seeded in 24 replicates in 2 sets of 96-well flat-bottom culture plates containing irradiated spleen cells (1 3 105 per well) under Th1 or Th17 polarizing conditions, with one set of plates containing an optimal dose of immunizing peptide (10 mg/ml). Based on preliminary limiting dilution assay (LDA) estimates of frequencies, the number of T cells seeded in each well varied from 3 3 103 to 2 3 105. After 44 h of incubation, the plates were pulsed with 0.5 mCi [3H]thymidine/well for 6 h, harvested, and assessed for isotope incorporation. Positive microcultures were defined as those in which the level of incorporated thymidine exceeded the mean level in control cultures (no responders) by more than three SDs. The frequency of responder T cells was obtained by estimates of precursor frequency calculated using a program developed to analyze LDA data (29–31) that uses the Poisson distribution to calculate the frequency of responder T cells with 99% confidence limits.

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ENVIRONMENTAL FACTORS MODULATE THE EFFECT OF ADENOSINE Comparison of the effect of selective and nonselective AR agonists Because NECA nonselectively binds to all four types of ARs, we examined whether the effect was mediated via a specific AR. Groups of B6 mice were immunized with IRBP/CFA and either left untreated or injected on day 0 or 7 with an agonist specific for A2ARs (CGS 21680, 1 mg/kg body weight), A2BRs (BAY 60-6538), or the nonselective NECA; then on day 13, serum IL-17 levels (Fig. 3A), the percentage of proliferating IL-17+ cells among the in vitro–stimulated T cells (Fig. 3B, 3C), and IL-17 production by the responder T cells after in vitro stimulation (Fig. 3D) were measured. The results showed that the A2AR agonist (CGS21680) and NECA had a similar inhibitory effect on Th17 responses compared with the nonagonist-treated group in the early-treatment group, but a stimulatory effect in the late-treatment group. In contrast, the A2BR agonist caused a moderate increase in the Th17 responses regardless of the time of treatment, suggesting that the nonselective agonist NECA mainly acts via A2ARs, which can exert either an enhancing or an inhibitory effect on the Th17 response. Differential effects of an AR agonist on Th1 and Th17 responses To determine whether AR agonists have the same or a different effect on different types of responder T cells, we also examined the effect of NECA on Th1 responses. First, we measured serum IFN-g levels at day 13 in immunized mice with or without early or late NECA treatment. As shown in Fig. 4A, both sets of NECA-treated mice produced significantly less IFN-g and, as shown in Fig. 4B, the frequency of in vivo primed IFN-g–producing cells was also significantly decreased in both sets of NECA-treated mice. After stimulation with the immunizing peptide under Th1-polarized conditions in vitro, IFN-g production (Fig. 4C) and the number of IFN-g–expressing cells (Fig. 4D) were both significantly

FIGURE 3. Comparison of the effects of a nonselective AR agonist (NECA) and specific A2AR or A2BR agonists. Groups of B6 mice were immunized with IRBP/CFA, then were either left untreated or were injected on the day of immunization or 7 d later with NECA (nonselective; 5 mg/kg), CGS 21680 (A2AR-specific agonist, 1 mg/kg), or BAY 60-6538 (A2BR agonist, 1 mg/kg) as indicated. All the mice were euthanized on day 13; then serum IL-17 levels were measured (A) and the relative number of in vitro primed, IL-17+ IRBP-specific Th17 responders compared with that in the untreated group (control) measured by LDA (B), and in vitro differentiation toward Th17 cells (C) and the production of IL-17 by in vivo primed T cells after in vitro antigenic stimulation (D) assessed as described in Fig. 1. Except in (C), the results are the mean 6 SD for one study using four mice, and the experiment was repeated four to five times with similar results. **p , 0.01.

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mice, being either inhibited or enhanced, depending on the time point when NECA was injected. Fig. 1A shows that serum IL-17 levels were reduced by early NECA treatment and increased by late treatment. Intracellular staining of in vitro–stimulated, IRBPspecific T cells showed that late treatment resulted in a significantly higher percentage of IL-17+ ab T cells (Th17 response) than in mice not treated with NECA, whereas early treatment resulted in a significantly depressed Th17 response compared with control mice (Fig. 1B, 1C). Measurement of secreted IL-17 levels showed that responder T cells from late-treatment mice produced much higher levels of IL-17 than T cells from non–NECA-treated mice, whereas cells from early-treatment mice produced minimal amounts of IL-17 (Fig. 1D). We also performed LDA, which measures the frequency of in vivo primed Ag-specific Th17 cells before in vitro expansion (25, 29, 34). As shown in Fig. 1E, the frequency of in vivo primed IL-17+ IRBP1–20–specific T cells was significantly increased in late-treatment mice (.40 per 100,000 responder T cells compared with 22 in controls) and significantly decreased in early-treatment mice (,5 per 100,000 responder T cells). We also compared the pathogenic activity of the Th17 polarized cells derived from the different groups of mice. Our results showed that the number of IRBP1–20–specific Th17 cells in late-treatment mice was significantly higher (2.5 3 106/mouse) than that in non–NECA-treated mice (1.0 3 106/mouse) and was reduced in early-treatment mice (0.4 3 106/mouse; Fig. 2A). Moreover, as shown by funduscopy (Fig. 2B) and pathological examination (Fig. 2C), when 1.5 3 106 activated IRBP-specific T cells were adoptively transferred into naive mice by i.p. injection, the induced EAU was significantly more severe using cells from late-treatment mice than using cells from control mice, whereas no disease was seen using cells from the early-treatment group, demonstrating that the late-treatment mice generated more IL-17+ pathogenic T cells.

The Journal of Immunology

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decreased in both sets of NECA-treated mice, although late treatment had less of an inhibitory effect. These results contrast markedly with the effect on the Th17 responses shown in Fig. 1. Effect of an AR is modulated by gd T cells We recently reported that activation of gd T cells plays a key role in the activation and expansion of a Th17 autoimmune response (22–25). To determine whether the effect of an AR agonist on

Th17 autoreactive T cells involved gd T cells, groups (n = 4) of TCR-d2/2 mice were immunized with IRBP1–20/CFA and were left untreated or underwent early or late treatment with NECA. As shown in Fig. 5A, on day 13, both sets of NECA-treated TCR-d2/2 mice had significantly lower serum levels of IL-17 than the non– NECA-treated group. Likewise, both NECA-treated groups had lower numbers of in vivo primed IRBP1–20–specific Th17 cells as detected by LDA (Fig. 5B), a lower percentage of IL-17+

FIGURE 5. Enhancing effect of late NECA treatment on the Th17 response requires gd T cells. Twelve TCR-d2/2 mice were immunized with IRBP/ CFA and then separated into three groups, which were left untreated or received early or late NECA treatment, then were tested at day 13 postimmunization. Results shown are the mean 6 SD for one study using four mice, and the experiment was repeated four to five times with similar results. (A) Serum IL-17 levels determined by ELISA, (B) frequency of in vivo primed Th17 cells by LDA, (C) percentage of IL-17+ cells among the in vitro Agstimulated, IRBP-specific T cells assessed by cytoplasmic staining, and (D) in vitro cytokine production by the responder T cells determined by ELISA. **p , 0.01.

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FIGURE 4. Effects of NECA on Th1 responses are different from those on Th17 responses. The Th1 responses were evaluated in the same way as described for Th17 responses in Fig. 1. Except in (D), results are the mean 6 SD for one study using four mice and the experiment was repeated four to five times with similar results. (A) Serum IFN-g levels assessed by ELISA. (B) LDA assay of the frequency of IFN-g–producing cells among responder T cells. (C) IFN-g levels in the culture supernatants of T cells after exposure to immunizing Ag and APCs. (D) Cytoplasmic staining of IFN-g+ cells among the in vitro Ag-stimulated IRBP-specific T cells. **p , 0.01.

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ab T cells in the in vitro Ag-stimulated T cells (Fig. 5C), and lower IL-17 production by in vitro Ag-stimulated T cells (Fig. 5D), showing that the enhancing effect of late treatment with an AR agonist on Th17 responder T cells requires a gd T cell response.

(5% of the total cell number) from immunized B6 mice that had been either left untreated or had been stimulated for 48 h in vitro with NECA. The results showed that NECA-treated gd T cells were much more effective than untreated gd T cells in increasing the percentage of IL-17+ ab T cells (Fig. 6C).

Discussion

To further examine a possible role of gd T cells in the immunomodulatory effect of NECA in EAU, we determined the effect of in vivo early or late injection of NECA on gd T cell activation in IRBP1–20–immunized B6 mice on day 13 after immunization. As shown in Fig. 6A, only 1.8% of total CD3+ splenic T cells in naive mice were gd T cells, whereas the corresponding numbers were 7.0% in non–NECA-treated immunized mice, 4.9% in immunized mice with early NECA treatment, and 10% in late NECA treatment mice. In addition, the percentage of gd T cells expressing the T cell activation molecule CD44 was 5.1% in naive mice and 32% in immunized non–NECA-treated mice, and this percentage declined to 18% with early NECA treatment and increased to 63% with late NECA treatment (Fig. 6A), paralleling the effect on the Th17 response. To determine whether the activation status of gd T cells had an effect on the enhancing effect of the AR agonist on Th17 response in immunized mice, we compared the effect of in vitro NECA treatment (100 nM) on gd T cells separated from naive and immunized mice. As shown in Fig. 6B, gd T cells isolated from naive mice produced little IL-17 after incubation for 48 h in medium with or without NECA, whereas gd T cells isolated from immunized mice produced moderate amounts of IL17 in the absence of NECA, and levels were significantly enhanced by culture in medium with NECA. Finally, we performed a study in which the percentage of IL-17+ T cells was measured among responder T cells from immunized TCR-d2/2 mice after 5 d of in vitro stimulation in the absence or presence of gd T cells

Previous studies have shown that adenosine can either inhibit or enhance an immune response, depending on which of the four identified ARs (35) is/are activated (13–20). In general, activation of A2ARs has anti-inflammatory effects (15, 16, 36, 37), whereas activation of A2BRs promotes inflammatory responses (11, 18, 38). However, this does not apply to all immune responses. For example, proinflammatory effects have been seen as a result of A2AR activation (11, 18), and anti-inflammatory effects of A2BR activation have been reported (11, 39–41). A study in A2AR2/2 mice showed that they developed more severe EAE than syngeneic B6 mice, but treatment of wild-type B6 mice with an A2AR antagonist inhibited, rather than enhanced, the development of EAE (21), suggesting that the mechanisms by which adenosine agonists/antagonists affect an immune response is more sophisticated than currently thought. Because AR-based treatments have been promoted for the treatment of autoimmune diseases (7–9) and neurological diseases (10) and in transplantation (42), clarification of the mechanisms involved will help determine how to use such molecules for immune modulation. Early studies investigating the effect of adenosine on T cell biology mostly examined the response of IFN-g–producing (Th1-type) T cells, so we were interested in determining whether AR agonists are also able to manipulate Th17 pathogenic autoimmune responses in EAU, and whether Th1 and Th17 autoreactive T cells respond similarly to AR agonists. Using our recently established system for studying the role of gd T cells in Th17 autoimmune responses (22– 25), we examined whether AR agonists exert their effect by acting

FIGURE 6. In vitro treatment with NECA promotes the enhancing effect of gdTCR+ T cells on Th17 autoreactive T cells. (A) Groups of B6 mice were left untreated or were immunized with IRBP1–20/CFA, then were left untreated or received early or late NECA injection, and all groups euthanized 13 d postimmunization and CD3+ T cells from spleen and draining lymph nodes were prepared; then the percentage of abTCR+ and gdTCR+ T cells among the separated CD3+ cells and the surface CD44 expression of gdTCR cells were determined after staining with anti-gdTCR and anti-abTCR Abs (left panels) or anti-CD44 and anti-gdTCR Abs (right panels), followed by FACs analysis. (B) gd T cells from immunized mice produce more IL-17 in response to in vitro treatment with NECA than gd T cells from naive mice. Pooled gd T cells isolated from naive or immunized B6 mice (n = 4) were incubated with 100 nM NECA for 48 h; then IL-17 levels in the culture supernatants were measured by ELISA. Results shown are the mean 6 SD for one study using four mice, and the experiment was repeated four to five times with similar results. **p , 0.01. (C) ab T cells from immunized TCR-d2/2 mice were stimulated in vitro for 5 d in the absence or presence of gd T cells (5% of total cell number) isolated from immunized B6 mice that had either been left untreated or had been incubated for 48 h with 100 nM NECA.

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gd T cells from immunized mice respond more vigorously to NECA than gd T cells from naive mice

The Journal of Immunology

In vivo, T cells are exposed to a complex of cytokines, TLR ligands, and adenosine metabolites, particularly in an inflammatory environment, and display considerable functional plasticity. Recent studies have shown that the activity of regulatory T cells can be reduced in response to environmental triggers, a process designated as “instability of regulatory T cell function” (44–47). Our results provide support for this by showing that, as one of the major regulatory T cells in the Th17 autoimmune response (22, 23, 25, 28), gd T cell function was also modulated by inflammatory factors. Adenosine is produced under various stress conditions (37, 48–51) and exerts multifaceted effects on various functions, including that of the immune system, and its effect on immune responses deserves further examination.

Disclosures The authors have no financial conflicts of interest.

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25.

ENVIRONMENTAL FACTORS MODULATE THE EFFECT OF ADENOSINE